In Vitro 5-Lipoxygenase Inhibition of Polyphenolic Antioxidants from Undomesticated Plants of South Africa

Full Length Research Paper

In vitro 5-Lipoxygenase inhibition of polyphenolic antioxidants from undomesticated plants of South Africa

Uma Sankar Akula and Bharti Odhav*

Durban University of Technology, Faculty of Engineering, Science and the Built Environment, Department of Biotechnology, ML Sultan Campus, P.O. Box 1334, Durban, 4000, South Africa.

Accepted 10 July 2008

Aqueous and methanolic extracts of 18 leafy vegetables from South Africa were investigated for their free radical scavenging activity, total phenolic content and anti-inflammatory properties. Amaranthus dubius, Amaranthus hybridus, Amaranthus spinosus, Asystasia gangetica, Bidens pilosa, Centella asiatica, Ceratotheca triloba, Chenopodium album, Cleome monophylla, Emex australis, Galinsoga parviflora, Justicia flava, Momordica balsamina, Oxygonum sinuatum, Physalis viscosa, Portulaca oleracea, Senna occidentalis and Solanum nigrum were used in this study. In the radical scavenging studies using 2,2-diphenyl-2-picrylhydrazyl hydrate(DPPH), methanolic extracts from six plants almost completely inhibited DPPH absorption (J. flava-96.6%, P. oleracea-96.5%, M. balsamina-94.7%, O. sinuatum-92.1%, S. nigrum-92.0% and A. hybridus-90.5%), whereas methanolic extracts from E. australis-78.6% and G. parviflora-76.5% showed lowest activities among the plants studied. Aqueous extracts were considerably less effective radical scavengers compared to methanolic extracts showing highest activity with J. flava-48.0% and lowest with A. gangetica-36.2%. A correlation between radical scavenging activity of extracts with total phenolic content was observed. Out of 18 plants studied for anti-inflammatory activity, methanolic extracts from eight plants showed significant inhibition of 5- lipoxygenase (5-Lox) activity. Among the plants studied Bidens pilosa (IC50 21.8 µg/ml) showed maximum anti-inflammatory activity while Emex australis (IC50 81.4 µg/ml) showed minimum inhibition of 5-Lox activity. Large amounts of phenolic compounds may contribute towards the antioxidant and anti-inflammatory properties among the plants studied.

Key words: Leafy vegetables, radical scavenging, DPPH, total phenolics, 5-lipoxygenase, anti-inflammatory.

INTRODUCTION

The uncontrolled production of oxygen free radicals and of ageing, such as cancer, cardiovascular disease, cata- the unbalanced mechanism of antioxidant protection racts, immune system decline and brain dysfunction result in the onset of many diseases and accelerate age- (Ames et al., 1990; Percival, 1998; Young and Woodside, ing. The antioxidants when present at low concentrations 2001). Fortunately, free radical formation is controlled compared to that of an oxidizable substrate, significantly naturally by various beneficial compounds known as anti- delays or inhibits the oxidation of that substrate (Percival, oxidants (Percival, 1998). 1998; Young and Woodside, 2001; Atoui et al., 2005). When the availability of antioxidants is limited, this The physiological role of free radical and hydroxyl free damage can become cumulative and debilitating oxide- radical-scavengers is to prevent damage to cellular com- tive stress results. Antioxidants are capable of stabilizing, ponents arising as a consequence of chemical reactions or deactivating free radicals before the latter attack cells involving free radicals. The key role for free radicals is as and biological targets. They are therefore critical for major contributors to ageing and to degenerative diseases maintaining optimal cellular and systemic health and well- being (Percival, 1998). Most antioxidants isolated from

higher plants are polyphenols, which show biological acti- *Corresponding author. E-mail: [email protected]. Tel: +27 31 vity as antibacterial, anti-carcinogenic, anti-inflammatory, 373 5330. Fax: +27 31 373 5351. anti-viral, anti-allergic, estrogenic and immune-stimulating 208 J. Med. Plant. Res.

Ing effects (Larson, 1988). matory activity of a drug (Alitonou et al., 2006). The antioxidant activity of phenolics is mainly due to Many of the plant anti-oxidants are polyphenols which their redox properties, which allow them to act as reduc- have carbon-based aromatic phenyl-ring compounds ing agents, hydrogen donors, and singlet oxygen quen- which are easily oxidized to quinones by reactive oxygen chers. In addition, they have a metal chelation potential. species, a property that helps account for their free radi- Synthetic anti-oxidants such as butylated hydroxyanisole cal scavenging capacity. The leaves of many traditional (BHA), butylated hydroxytoluene (BHT) and tertbutyl- leafy vegetables supplement the diets, which consist hydroquinone (TBHQ) have been used widely as anti- mainly of cereals and grains (Van Wyk and Gericke, oxidants in foods, but concerns over the safety of use 2000). Most of these plants lack comprehensive data that have led towards interests in natural anti-oxidants can corroborate the traditional knowledge. The aim of this (Wanasundara and Shahidi, 1998). These synthetic anti- work was to investigate 18 traditional leafy vegetables oxidants are substituted phenolic compounds, and subse- found in the Kwa-Zulu Natal province of South Africa for quently much of the research on natural anti-oxidants has their anti-oxidative and anti-inflammatory potential, and also focused on phenolic compounds, in particular the correlate this to total phenolic compounds. flavonoids and hydroxycinnamic acids (Martı´nez- Valverde et al., 2002). Phenolic compounds also possess an array of potentially beneficial lipoxygenase inhibitory MATERIALS AND METHODS and anti-oxidant properties; they have been used for the Plant material treatment of inflammatory diseases (Sreejayan and Rao, 1996). Eighteen traditional leafy vegetables were used in this study. They The mechanisms of inflammation involve a series of were collected from different locations in Kwa-Zulu Natal, South events in which the metabolism of arachidonic acid plays Africa. Table 1 lists the leafy vegetables and consumption practices an important role. Prostaglandins are involved in the used in this study with the following information: scientific name, complex process of inflammation and are responsible for family, English and Zulu names. Local floristic keys were used for determining the species. Data on different species and the Zulu the pain (Jager et al., 1996). It can be metabolized by the names have been sourced from publications on the flora of South cyclooxygenase (COX) pathway to prostaglandins and Africa (Odhav et al., 2007; Hutchings, 1996). Voucher specimens thromboxane A2, or by the lipoxygenase (LOX) pathway are housed in the Ward Herbarium, University of Kwa-Zulu-Natal, to hydroperoxy-eicosatetraenoic acids (HPETE’s) and Westville campus, Durban, South Africa. leukotrienes (LT’s), which are important biologically active mediators in a variety of inflammatory events Sample extraction (Alitonou et al., 2006, Piper et al., 1994). Upon appro- priate stimulation of neutrophils, arachidonic acid is The leaves (100 g) were dried at 30ºC in a oven (Memmert) and cleaved from membrane phospholipids and can be con- powdered in a Salton blender. Ten grams were dissolved in 200 ml verted to leukotrienes and prostaglandins through 5- of dd H2O/ 80% methanol (vol/vol) for one hour and filtered with a Whatman No. 1 filter paper. The filtrate was then concentrated to lipoxygenase(5-Lox) or cyclooxygenase(Cox) pathways 15 ml using a Buchi RE 121 Roto-evaporator including a Buchi 461 respectively (Bouriche et al., 2005). Furthermore, inflam- water bath set at a temperature of 50ºC. This concentrate was matory processes also involve reactive oxygen species freeze dried using a Virtis Benchtop Freeze drier. started by leucocyte activation. Grabmann et al. (2000) found that eucalyptus and myrtle essential oils attenuated DPPH radical scavenging assay leucocyte activation by scavenging hydroxyl radicals indirectly produced by leucocyte degranulation, thereby The radical scavenging activity of plant extracts was measured interfering with inflammatory processes by acting as using the stable free radical scavenger, DPPH (2, 2-diphenyl-2- antioxidants. Therefore, screening of antioxidant pro- picrylhydrazyl hydrate), decoloration assay described by Choi perties may provide important information about the (2002). The freeze dried plant samples (1.0 mg/ml) from each of the plant extracts were diluted in ethanol to give final concentrations of potential activity of a drug on inflammatory processes 500, 250, 100, 50 and 10 µg/ ml. One ml of a 0.3 mM DPPH in (Njenga and Viljoen, 2006) The establishment of new in ethanol solution was added to 2.5 ml of sample solution of different vitro test systems has stimulated the screening of plants concentrations and allowed to react at room temperature for 30 aiming to find leads for the development of new drugs. min. The resulting colour was measured at A518 spectropho- The plant lipoxygenase pathway is in many respects the metrically (Varion Cary I-E UV visible spectrophotometer). The equivalent of the ‘arachidonic acid cascades’ in animals radical scavenging activity was measured as the decolourization percentage of the test sample using the following formula: (Gardner, 1991). For this reason, the in vitro inhibition of soybean lipoxygenase constitutes a good model for the DPPH Scavenging capacity % =100 - [(Absorbance of sample - screening of plants with anti-inflammatory potential (Abad Absorbance of blank) x 100/Absorbance of control] et al., 1995). A combination of this assay with an eva- luation of the radical scavenging activity by the 1,1- While ethanol (1.0 ml) plus plant extract solution (2.5 ml) was used as a blank, DPPH solution plus ethanol was used as a negative diphenyl-1-picrylhydrazyl (DPPH) method (Choi, 2002) control. The positive controls were DPPH solution plus each 1 mM constitutes a good indication on the potential anti-inflam- Rutin. Akula and Odhav 209

Table 1. List of species and consumption practices of leafy vegetables in Kwa-Zulu Natal area of South Africa.

Family Scientific name English Name Zulu Name Consumption Acanthaceae Asystasia gangetica hunter’s spinach isihobo during famine Acanthaceae Justicia flava yellow justicia ipela occasionally Amaranthaceae Amaranthus dubius wild spinach imbuya regularly Amaranthaceae Amaranthus hybridus cockscomb imbuya regularly Amaranthaceae Amaranthus spinosus spiny pigweed imbuya regularly Apiaceae Centella asiatica marsh pennywort icudwane during famine Asteraceae Bidens pilosa black jack amalenjane regularly Asteraceae Galinsoga parviflora gallant soldier ushukeyana regularly Capparaceae Cleome monophylla spindle-pod isiwisa regularly Chenopodiaceae Chenopodium album fat hen imbikilicane regularly Cucurbitaceae Momordica balsamina balsam apple umkaka regularly Fabaceae Senna occidentalis coffee senna isinyembane during famine Pedaliaceae Ceratotheca triloba wild foxglove udonqabathwa regularly Polygonaceae Emex australis devil’s thorn inkunzane occasionally Polygonaceae Oxygonum sinuatum starstalk untabane during famine Portulacaceae Portulaca oleracea purslane madilika regularly Solanaceae Physalis viscosa sticky gooseberry uqadolo during famine Solanaceae Solanum nigrum black nightshade umsobosobo regularly

Estimation of total phenolic compounds in plant extracts such a manner that an aliquot of each (30 µl) yielded a final concentration of maximum 100 ppm in each assay. The initial The content of total phenolic compounds in plant methanolic reaction rate was determined from the slope of the straight line extracts was determined by Folin–Ciocalteu method (Miliauskas et portion of the curve and the percentage inhibition of the enzyme al., 2004). For the preparation of calibration curve 1 ml aliquots of activity was calculated by comparing with the control (using 30 µl of 0.024, 0.075, 0.105 and 0.3 mg/ml ethanolic gallic acid solutions phosphate buffer (pH 9.0) instead of 30 µl of the inhibitor solution). were mixed with 5 ml Folin–Ciocalteu reagent (diluted three-fold) Each inhibitor concentration was tested in triplicate and the results and 4 ml (75 g/l) sodium carbonate. The absorption was read after averaged; the concentration that gave 50% inhibition (IC50) was 30 min at 200ºC at 765 nm and the calibration curve was drawn. calculated from the outline of the inhibition percentages as a One ml methanolic plant extract (10 g/l) was mixed with the same function of the inhibitor concentration (Njenga and Viljoen, 2006; reagents as described above, and after 1 h the absorption was Baylac, 2003). Aqueous extracts (IC50 ≥100 µg/ml) were not taken measured. Total content of phenolic compounds in plant methanol in this study. A negative result in the lipoxygenase assay does not extracts in gallic acid equivalents (GAE) was calculated by the necessarily mean that the plant is without anti-inflammatory activity. following formula: The active compounds could work at other sites in the complex process of inflammation (Jager et al., 1996). C = c.V/m

Where: C = total content of phenolic compounds (mg/g) plant Statistical analysis extract in GAE; c = the concentration of gallic acid established from the calibration curve (mg/ml); V = the volume of extract in ml; and m All the analysis were carried out in triplicate and the results were = the weight of pure plant methanolic extract in g. expressed as the mean ±SD. Correlation co-efficient (R) to determine the relationship between two or more variables among Radical Scavenging activity tests and content of total phenolic Determination of the 5- lipoxygenase activity compounds were calculated using MS Excel software (CORREL statistical function). Regression analysis was used to calculate IC50, Lipoxygenase is known to catalyse the oxidation of unsaturated defined as the concentration of inhibitor necessary for 50% fatty acids containing 1-4 diene structures. The conversion of inhibition of the enzyme reaction. linoleic acid to 13- hydroperoxy linoleic acid was followed spectro- photometrically by the appearance of a conjugate diene at 234 nm on a UV/visible spectrophotometer (Varion Cary 1E UV- Visible RESULTS AND DISCUSSION spectrophotometer). Nordihydroguaiaretic acid (NDGA) and Rutin known inhibitors of soybean lipoxygenase, were used as controls. For all the plants the methanolic extracts were more The reaction was initiated by the addition of aliquots (50 µl) of a effective DPPH radical scavengers than the aqueous soybean lipoxygenase solution (prepared daily in potassium extracts (Table 2). Almost complete DPPH absorption phosphate buffer M pH 9.0) in a sufficient concentration to give an was found with methanolic extracts from Justicia flava easily measurable initial rate of reaction to 2.0 ml of sodium linoleate (100 µM) in phosphate buffer; the enzymatic reactions (96.6%), Portulaca oleracea (96.5%), Momordica balsa- were performed in absence or in presence of inhibitor and their mina (94.7%), Oxygonum sinuatum (92.1%), Solanum kinetics were compared. The inhibitors were dissolved in DMSO in nigrum (92.0%) and Amaranthus hybridus (90.5%). 210 J. Med. Plant. Res.

Table 2. Antioxidant values of the plant extracts among leafy vegetables from Kwa-Zulu Natal, South Africa.

Percent scavenging activity Percent scavenging activity Plant name of methanolic extract (a, b) of aqueous extract(a, b) Amaranthus dubius 78.4±0.32 42.6±0.14 Oxygonum sinuatum 92.1±0.26 44.1±0.25 Asystasia gangetica 84.7±0.38 36.2±0.18 Galinsoga parviflora 76.5±0.44 42.9±0.22 Physalis viscosa 82.4±0.30 40.7±0.12 Justicia flava 96.6±0.54 48.0±0.08 Amaranthus spinosus 88.2±0.22 40.6±0.42 Solanum nigrum 92.0±0.18 42.1±0.36 Amaranthus hybridus 90.5±0.24 42.0±0.12 Momordica balsamina 94.7±0.36 42.5±0.32 Centella asiatica 88.2±0.12 39.0±0.24 Chenopodium album 82.5±0.38 40.8±0.18 Emex australis 78.6±0.22 39.2±0.26 Senna occidentalis 82.4±0.26 40.3±0.14 Ceratotheca triloba 84.9±0.16 36.7±0.28 Bidens pilosa 88.7±0.34 40.5±0.38 Cleome monophylla 84.7±0.26 36.4±0.26 Portulaca oleracea 96.5±0.38 44.3±0.08

aMean values obtained from experiments performed in triplicate. bMean value determined graphically and standard deviation.

Others such as Emex australis (78.6%) and Galinsoga from eight plants showed significant inhibition of 5- parviflora (76.5%) had lower activities. The aqueous ex- lipoxygenase (5-Lox) activity (Table 4). Among the plants tracts were considerably less effective radical scavengers studied Bidens pilosa (IC50 21.8 µg/ml) showed maximum showing highest activity with J. flava (48.0%) and lowest anti-inflammatory activity while Emex australis (IC50 81.4 with Asystasia gangetica (36.2%). µg/ml) showed minimum inhibition of 5-Lox activity. An The content of phenolic compounds (mg/g) in metha- interesting finding from this study shows that the mem- nolic extracts (Table 3) expressed in GAE, varied bet- bers of family Asteraceae have high levels of anti-inflam- ween 21.1 and 42.4. The highest amounts were found in matory activity [(Bidens pilosa (IC50 21.8 µg/ml), Galin- the extracts of A. hybridus (42.4) followed by J. flava soga parviflora (IC50 30.7 µg/ml) whereas members (40.6). Furthermore, plants showing high radical sca- from family Solanaceae venging activity [J. flava (96.65%) and M. balsamina S. nigrum (IC50 27.1 µg/ml), P. viscosa (IC50 29.5 (94.7%)] also contain high amounts of phenolic com- µg/ml)] show moderate activity and those from Amaran- pounds (40.6 and 39.8 respectively). A positive corre- thaceae like A. hybridus (IC50 77.2 µg/ml), A. dubious 2 lation (R = 0.78) was found, suggesting that the anti- (IC50 69.4 µg/ml) and A. spinosus (IC50 57.3 µg/ml) oxidant capacity of the methanolic plant extracts is due to showed minimum inhibition of 5-Lox activity. Earlier a great extent to the polyphenol content. The quantitative studies (Njenga and Viljoen, 2006; Abajo et al., 2004; correlation between the antioxidant activity and the Matu and Van Staden, 2003) found similar results with content of polyphenols in most of the plants (Figure 1) some members of the Asteraceae family, whereas O. indicates that phenolic compounds are likely to contribute sinuatum (IC50 32.1 µg/ml), E. australis (IC50 81.4 µg/ml) to the radical scavenging activity among these plant from Polygonaceae and J. flava (IC50 31.4 µg/ml), A. extracts. gangetica (IC50 55.0 µg/ml) from Acanthaceae showed Higher radical scavenging activities of polyphenolic marked variance in 5-Lox inhibition. antioxidants can be ascribed to chemical reactions fol- Antioxidants are known to inhibit plant lipoxygenases lowing their oxidation (Hotta et al., 2002) and studies on (Ammon et al., 1993). Studies have implicated the antioxidant and anti-inflammatory activities of methanolic oxygen free radicals in the process of inflammation and extracts from Ledum groenlandicum (Dufour, 2007) have phenolic compounds may block the cascade process of indicated a possible correlation between the antioxidant arachidonic acid metabolism by inhibiting lipoxygenase activity and the phenolic compounds. Out of 18 plants activity, and may serve as a scavenger of reactive free studied for anti-inflammatory activity, methanolic extracts radicals which are produced during arachidonic acid me- Akula and Odhav 211

Table 3. Amount of total phenolic compounds (mg/g) in gallic acid equivalents among leafy vegetables from Kwa-Zulu Natal, South Africa

Plant name Total phenolic compounds, (mg/g) in Total phenolic compounds, methanolic extract (a,b) (mg/g) in aqueous extract (a.b) Amaranthus dubius 26.2±0.34 18.1±0.24 Oxygonum sinuatum 39.4±0.14 24.6±0.18 Asystasia gangetica 28.3±0.26 16.5±0.22 Galinsoga parviflora 24.1±0.30 15.3±0.36 Physalis viscosa 32.0±0.41 19.6±0.30 Justicia flava 40.6±0.38 26.2±0.16 Amaranthus spinosus 32.3±0.14 21.8±0.41 Solanum nigrum 37.6±0.22 16.4±0.31 Amaranthus hybridus 39.6±0.32 28.4±0.08 Momordica balsamina 39.8±0.54 25.2±0.44 Centella asiatica 31.8±0.38 16.1±0.28 Chenopodium album 26.8±0.12 15.4±0.22 Emex australis 27.4±0.36 20.8±0.18 Senna occidentalis 27.6±0.12 21.6±0.42 Ceratotheca triloba 35.2±0.44 14.6±0.24 Bidens pilosa 38.1±0.08 24.4±0.32 Cleome monophylla 32.6±0.20 12.5±0.18 Portulaca oleracea 37.6±0.14 23.6±0.20

aMean values obtained from experiments performed in triplicate. bMean value determined graphically and standard deviation.

Table 4. Anti-inflammatory activity (5-Lipoxygenase 50 inhibition) of methanolic extracts among leafy R2 = 0.7874 vegetables from Kwa-Zulu Natal, South Africa. t 45 n e t

n Plant name 5-Lox IC50 (µg/ml)* o 40 c Amaranthus dubius 69.4 c i l

o Oxygonum sinuatum 32.1

n 35 e

h Asystasia gangetica 55.0 p

l 30

a Galinsoga parviflora 30.7 t o

T Physalis viscosa 29.5 25 Justicia flava 31.4

20 Amaranthus spinosus 57.3 70 75 80 85 90 95 100 Solanum nigrum 27.1 Amaranthus hybridus Total anti oxidant activity 77.2 Momordica balsamina 40.3

Figure 1. Correlation between total anti-oxidant activity and Centella asiatica 38.6 total polyphenol content of methanolic extracts of 18 leafy Chenopodium album 33.2 vegetables from Kwa-Zulu Natal, South Africa. Emex australis 81.4

Senna occidentalis 43.5 Ceratotheca triloba 56.1 metabolism (Trouillas et al., 2003; Sreejayan and Rao, Bidens pilosa 21.8 1996). The anti-inflammatory activities of the eighteen Cleome monophylla 32.0 plant extracts could be explained by the potent inhibitory Portulaca oleracea 37.9 effects of their phenolic compounds on arachidonic acid NDGA 4.1 metabolism through the lipoxygenase pathway. It is Rutin 7.3 possible that the correlation observed between the three tests is due to the considerably high amounts of phenolic *Inhibition (%) of 5-lipoxygenase activity obtained and IC50 values represent the mean ±S.D. of three compounds among the plants studied. determinations. 212 J. Med. Plant. Res.

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